When mitochondrial respiration is compromised, the F₁F_o-ATP synthase reverses and consumes ATP, serving to maintain the mitochondrial membrane potential (Δψ_m). This process is mitigated by IF₁. As little is known of the cell biology of IF₁, we have investigated the functional consequences of varying IF₁ expression. We report that, (1) during inhibition of respiration, IF₁ conserves ATP at the expense of Δψ_m; (2) overexpression of IF₁ is protective against ischemic injury; (3) relative IF₁ expression level varies between tissues and cell types and dictates the response to inhibition of mitochondrial respiration; (4) the density of mitochondrial cristae is increased by IF₁ overexpression and decreased by IF₁ suppression; and (5) IF₁ overexpression increases the formation of dimeric ATP synthase complexes and increases F₁F_o-ATP synthase activity. Thus, IF₁ regulates mitochondrial function and structure under both physiological and pathological conditions.